Although ALS was first described in 1869, the mechanisms involved in the pathogenesis of this neurodegenerative disease still remain largely unknown. Several mechanisms that are not mutually exclusive have been proposed. These include oxidative stress, glutamate-induced excitotoxicity, cytoskeletal abnormalities, protein aggregation, mitochondrial dysfunction, and, more recently, inflammation. The role of inflammation and microglia in ALS and other CNS diseases is currently a matter of great debate and controversy. Although numerous cytokines are up-regulated in microglia of mice expressing mutant SOD1 transgenes, deletion in the gene encoding IL-1β and TNF does not change the outcomes of the diseases (Nguyen et al., 2001
; Gowing et al., 2006
). However, SOD1G37R
mice that had MyD88-competent BMDM developed the disease later, survived longer, and had less neurodegeneration than those that were transplanted with MyD88-deficient BM cells. These data suggest a novel neuroprotective role of competent BMDM in a mouse model of ALS.
The discovery that ~20% of familial ALS cases are caused by mutations in SOD1 has enabled the development of animal and cell culture models and led to much of our current understanding of the neurodegenerative mechanisms in ALS. SOD1 is a ubiquitously expressed protein, which protects cells from damage by free radicals. ALS often starts focally. It is still unclear how the toxicity of mutant SOD1 is propagated from one localized group of cells to another. Based on the recent evidence that extracellular mutant SOD1 proteins could be selectively secreted and trigger microgliosis and neuronal death in cultured cells (Urushitani et al., 2006
), we injected WT SOD1 and mutant proteins into the mouse CNS. G93A mutant protein was used in our experiments because it was the only SOD1 mutant protein available to us. In agreement with this study, our data demonstrate that mutant SOD1 stimulates inflammation and recruitment of BMDM. WT SOD1 protein was used as a control to ascertain that these effects were specific to the mutant protein and not to potential traces of endotoxin. The inflammatory response caused by G93A is largely dependent on MyD88 signaling but it is still not known whether this protein binds to specific immune receptors in microglia. However, G93A is able to stimulate TNF production from the microglial cell line (Urushitani et al., 2006
) and an acute intracerebral infusion of this protein is not neurotoxic. We therefore suggest that the inflammatory properties of mutant SOD1 are directly mediated by the MyD88 pathway in microglia and not via other products released by dying cells.
Previous studies have shown that in chimeric SOD1G93A
mice, BM-derived cells differentiate into microglia in the spinal cord and brain. In addition, it has been suggested that the number of GFP-positive cells in the spinal cord is associated with disease progression (Corti et al., 2004
; Solomon et al., 2006
). In this paper, we generated chimeric SOD1G37R
mice and confirmed the extensive distribution of GFP-positive cells throughout the brain and spinal cord in both mouse models of ALS. In accordance with the previous studies, we observed few GFP-positive cells in the CNS before the disease onset (Fig. S1), but the number of GFP cells greatly increased during disease progression (Table S1, available at http://www.jcb.org/cgi/content/full/jcb.200705046/DC1
). More importantly, transplantation of BM from GFP- expressing mice does not affect disease progression of either SOD1G93A
mice when compared with their respective nonirradiated SOD1 groups. In contrast, transplantation of MyD88-deficient BM cells dramatically changed the disease onset and progression only in mice that express human mutant G37R.
The rationale to investigate the role of this adaptor protein was based on our initial observation that the MyD88 pathway mediates microglial activation and infiltration induced by mutant SOD1. The results from the chimeric mice suggest that BMDM acts as a natural defense mechanism against secreted mutant SOD1. Indeed, MyD88−/− BM transplantation led to the earlier disease onset and shorter lifespan of SOD1G37R mice compared with mice that received GFP cells. GFP–SOD1G37R and nonirradiated SOD1G37R mice were used as controls to exclude the possibility that these effects were attributable to irradiation. Histological analysis revealed a significant motor neuron and axon loss in MyD88−/−– SOD1G37R mice compared with the GFP–SOD1G37R and MyD88−/−–WT mice at the same age, which explains the intriguing earlier disease onset and death of MyD88−/−– SOD1G37R mice. We also found a more robust innate immune reaction in the spinal cord of these mice.
It has been shown that WT nonneuronal cells delayed disease onset by a mean of 1.2 mo for SOD1G37R
chimeras and extended their survival by 1.1 mo (Clement et al., 2003
). A recent study demonstrated that substitution of WT microglia for SOD1G93A
-expressing microglia prolonged the survival and disease duration of SOD1G93A
mice but had no effect on the onset (Beers et al., 2006
). However, reduced levels of mutant SOD1 in microglia did not change onset and the early disease phase but clearly slowed later paralysis (Beers et al., 2006
; Boillee et al., 2006
). These data do not necessarily contradict the experiments using MyD88-deficient mice. Indeed, the disease progression may be influenced by the levels of extracellular mutant SOD1 that resident microglia contribute to this extracellular pool. This may be an explanation for the neuroprotective properties of SOD1-deficient microglia and such effects may not be associated with the immune functions of these cells.
We have previously reported the existence of different populations of microglia that may have somewhat opposite roles. The double-edged sword of these cells that has been intensively reviewed in the past few years may also depend on the origin of microglia in the adult CNS. BMDM are very efficient in restricting amyloid deposits in a mouse model of Alzheimer's disease, whereas their resident counterparts seem unable to phagocyte this toxic protein (Simard et al., 2006
). The results from this paper support this concept and imply that impairment of such natural function of BMDM accelerates the neurodegenerative properties of secreted mutant SOD1. It is interesting to note that transplantation of MyD88-deficient BM cells did not change onset or survival of mice expressing human SOD1G93A
. These mice reach paralytic endstage ~2 mo after BM transplantation. The hematopoietic system takes 7–9 wk to be fully restored after lethal irradiation and the percentage of GFP- or MyD88-deficient cells is low during the first 4 wk of the chimera. This may explain why these cells are unable to modulate the disease progression in such an early onset model of neurodegeneration. This is not the case in mice expressing SOD1G37R
because restoration of BMDM is completed in these animals several months before the first symptomatic signs. The early onset may therefore explain why MyD88−/−
BM cells failed to change the mean life expectancy of SOD1G93A
Because of the potent beneficial role of WT BMDM in SOD1G37R
mice, the lack of significant difference in the disease onset and lifespan between G37R+/−
groups of mice may seem surprising. However, histological analysis revealed that microglial cells seem to be much more activated in G37R+/−
compared with G37R+/−
mice at the end stage of the disease (). In addition, at the end stage, G37R+/−
mice lost significantly more motor neurons than G37R+/−
mice (), which suggests that the context of MyD88 deficiency does affect these SOD1G37R
mice. Neuroprotection is not always associated with a significant extension of survival in mouse models of ALS. A recent study has shown that sodium valproate, the histone deacetylase inhibitor, exerts neuroprotective effects both in vitro and in vivo but it does not improve the survival of SOD1G86R
mice (Rouaux, et al., 2007
It is also important to mention that G37R+/−;MyD88−/− mice were much smaller than their littermates based on weekly body weight. In addition, the numbers of pups per carriage was lower in G37R+/−;MyD88−/− mice, with a mean of 4.5 compared with 7 pups from SOD1, 6 from MyD88−/−, and 8 from WT mice. Moreover, we were not able to generate a single G93A+/−;MyD88−/− mouse after >1 yr of crossbreeding between SOD1G93A and MyD88-deficient mice. We have not been able to generate homozygote MyD88−/− mice in using another mouse model of brain disease. Indeed, for >2 yr, we have attempted to breed amyloid precursor protein (APP)/presenelin 1 transgenic mice with our MyD88−/− colony and we have obtained only APP;MyD88+/− but not a single APP;MyD88−/− mouse. This indicates that these MyD88 homozygotes are not viable in the presence of highly toxic proteins, such as G93A and APP/presenelin 1. Compared with SOD1G93A, the lower level of SOD1G37R may also explain why we were able to generate G37R+/−;MyD88−/− mice.
Although unexpected, it is not surprising that the blockage of the MyD88-dependent intracellular signaling pathway in SOD1 mice does not have a marked influence on the survival. Indeed, previous studies have shown that exposure of MyD88−/−
macrophages with lipopolysaccharide results in the delayed activation of nuclear factor κB and MAPKs, which suggests the existence of a MyD88-independent signaling pathway (Kawai et al., 2001
). The paradoxical results from chimeric mice (where MyD88-deficient BMDM reduced survival) and crossed mice (where absence of MyD88 did not affect disease course) are best explained by putative compensatory mechanisms activated by developmental absence of MyD88. In fact, the strategies are very different. G37R+/−
mice may bear the compensation throughout their life, otherwise they will not be viable. This is obviously not the case in both models of transgenic SOD1 mice transplanted with BM-deficient cells because the MyD88 gene is present during all the developmental stages and is deleted only in BM stem cells at the age of 2 mo.
In summary, our data demonstrate a critical effect of MyD88 within BMDM in a mouse model of ALS disease, which suggests a novel neuroprotective role of BMDM and supports the protective functions of microglia at the early stage of ALS disease. With further studies, we hope to find a novel approach to optimize the neuroprotective role of BMDM in ALS disease.